Ribosomes are large molecular machines that catalyze the synthesis of new proteins. When multiple ribosomes recruit a mRNA, they form a polyribosome1,2. Only recently, scientists have tried to unravel the supramolecular structural and functional complexity of this ‘ensemble’3-9 (i.e. the assembly of nucleic acids and proteins). The crucial role of polyribosomes in the regulation of gene expression is highlighted by the discovery of many cancer-related mutations affecting ribosomal proteins, initiation factors as well as elongation factors interacting with polyribosomes10. Many components of the translation machinery are nowadays considered strategic target for the treatment of cancer and other pathologies (ribosomopathies)11. On top of that, recent discoveries have shown that stress conditions12 and drug treatment13 can specifically alter mRNAs associated within polyribosomes. All these evidences are underlining the pivotal role of polyribosomes in the process of protein synthesis and disease conditions. Additionally, the increasing number of gene expression studies on plants14, yeasts15 and mammalian cells16 concerning the recruitment of RNAs on polyribosomes (translatome) are emphasizing the importance of polyribosomes in gene expression regulation.
Unfortunately, existing tools and methods to investigate translation still need to be upgraded, and innovative methods to isolate and investigate the translatome and the complexity of polyribosomes are needed. Current protocols are expensive, laborious and not enough accurate (i.e. not able to capture only native, active polyribosomes) to dissect the post-transcriptional and translational processes between transcription and protein variation. A possible approach to gain information on translation is represented by the expression of affinity-tagged ribosomal proteins whose expression is controlled by tissues specific promoters. Tagged ribosomes (and the associated mRNAs) can be purified by affinity purification (RAP or translating RAP, TRAP)17. This method requires a specific mouse model and/or specific gene constructs; a long path that ends up in poorly versatile systems. The advance in sequencing and proteomics technology inspired the development of high-resolution (single nucleotide or single peptide) ‘omic’ techniques to profile translation, including the deep sequencing of ribosome protected fragments (ribosome-profiling)18, the global and quantitative profiling of initiating ribosomes (GTI-seq, QTI-seq)19,20 or the genome wide quantification of the newly synthetized proteome (Punch-P and pSILAC)21,22. Ribosome-profiling has been also used in combination to TRAP (translating ribosome affinity purification using genetically engineered organisms) to map the translatome under oxygen deprivation23, while Punch-P and pSILAC are complementary technologies to study gene expression regulation at the protein level. Besides different biological questions covered by these various approaches, all these techniques usually require the semi-quantitative analysis of mRNAs and proteins associated to (or produced by) polyribosomes24.
The isolation of polyribosome by ultracentrifugation in linear sucrose gradients, a technique in use from the 1960s, is the current ‘gold standard’ for gene expression translational studies. Messenger RNAs associated to polyribosomes are separated from the unbound-RNAs, the small (40S), the large (60S) ribosomal subunits and the 80S monosomes. RNA and proteins can be extracted from the gradient and analyzed by RT-qPCR, RNA-seq or by immunoblotting and mass spectrometry. Although this protocol is relatively cheap (˜70 € per sample) and paved the way for translatome studies25,26, it has some drawbacks. First, the technique requires expensive equipment, handling experience and is time consuming (˜6 hours). Second, sensitivity: large samples amounts (>105 cells) are required for a detectable signal profile during fractions' collection. Third, contaminations: although the presence of inactive polyribosomes is still under debate, it is known that at least in neurons polyribosomal fractions can contain non-translating (i.e. not active in protein synthesis) polyribosomes27,28. Furthermore, sucrose fractions could be marginally contaminated by other high molecular weight complexes4.
Therefore, a simple, fast, more accurate and cheap technique would have a strong impact on all gene expression studies.
Innovative approaches in this direction cannot disregard to include a detailed understanding of the ribosome structure and function. Eukaryotic ribosomes are ˜40% heavier than their bacterial counterparts and comprise two subunits (60s and 40S), four ribosomal RNAs and 79 ribosomal proteins, for a total mass of 4.5 MDa29. Ribosomes contain three active sites located in the large subunit, designated as A, P and E sites. The A site hosts an aminoacyl-tRNA (aatRNA), the P site a peptidyl-tRNA and the E site allows the free tRNA to exit the ribosome. The substrates of the reaction catalyzed by the large subunit are the incoming aminoacyl-tRNA in the A-site and the peptidyl-tRNA in the P-site. The reaction occurs in the peptidyl transferase centre (PTC). The α-amino group of the aa-tRNA attacks the carbon of the carbonyl, acylating the 3′-hydroxyl group of the peptidyl-tRNA; this resolves to yield a peptide extended by one amino acid esterified to the A site-bound tRNA and a deacylated tRNA in the P-site. Then, the ribosome translocates one codon forward.
The eukaryotic ribosome is the target of many small molecule acting as translation inhibitors. A large fraction of these molecules preferentially target the PTC30. This region is the catalytic core of the ribozyme (i.e. a catalytic active ribosome)31; and shares high-structural phylogenetic conservation with respect to the surrounding ribosomal areas32,33. Moreover, it is formed by RNA structural elements essential for peptide bound formation. Among small-molecular inhibitors, puromycin (an aminonucleoside antibiotic34) is an analogue of the 3′-end tyrosylated-tRNA, and is able to inhibit the ribosomal catalytic activity35 in both the prokaryotic and the eukaryotic ribosomes, binding the symmetrical V-shaped cavity that forms the PTC centre31,36 by entering into the A site. This drug played a central role in biochemical experiments aimed at understanding the mechanism of peptide-bond formation36-40. More recently, because of its ability to bind ribosomes and to be efficiently incorporated in the polypeptide nascent chain, puromycin has been extensively used as a tool to assay protein synthesis functions by means of radioactive puromycin41 or antipuromycin antibodies42. Additionally, puromycin has also been used to chemically link an mRNA to its coded protein43,44. These methods are based on the irreversible reaction of the α-amino group of puromycin with the carbon on the carbonyl, acylating the 3′ hydroxyl group of the peptydil-tRNA. This reaction resolves to yield a terminal puromycilated peptide, because the puromycin's amide cannot be cleaved. After that, protein synthesis stops. Given its mechanism of action, puromycin is incorporated in the nascent chain after reaction of its α-amino group.